Recently, Drs. Randall Bateman and David Holtzman at the Washington University School of Medicine developed a novel stable isotope labeling kinetics (SILK) methodology for measuring the metabolism of amyloid beta (AB) in the human central nervous system (CNS). This methodology measures the ratio of labeled to unlabeled AB at multiple time points after subject infusion with a stable isotope labeled amino acid. In this tye of study, as new human proteins are synthesized, they will incorporate the labeled amino acid. The changes in the ratio of labeled to unlabeled protein over time can be measured using a mass spectrometer and can be used to investigate the metabolism of proteins of interest. C2N has successfully commercialized this technology and is currently expanding the SILK assay to encompass production and clearance rates of multiple AB isoforms in an ongoing NIH-funded study. In addition, C2N is expanding the SILK method to include stable isotope spike absolute quantitation (SISAQ), enabling a novel method for calculating the concentration of the protein of interest. For this grant, we propose to expand the SILK and SISAQ methodologies to include measurement of the metabolism and concentration of the protein Tau in cerebrospinal fluid (CSF). The SILK-Tau assay would be of prime interest for use in clinical trials of novel Tau modulating drugs. It would allow drug makers to assess the effect that their drugs have in the human brain in early stage studies. In addition to measuring metabolism of Tau using the SILK assay, we believe that the SISAQ assay has both complementary and standalone value. Presently, the only available analytical methods for measuring Tau concentrations in biological fluids are antibody based ELISA assays. Tau concentration measurements by ELISA are known to vary widely from lab to lab and from study to study with relatively high variances reported both within and between sites. Based on our preliminary experience with the SISAQ-AB method, the variability of the SISAQ assay is significantly lower than what we see in ELISA assays. Phase I of this grant will focus on development of methods that will allow us to measure Tau metabolism and concentration using the SILK and SISAQ assays. In order to confirm that the assays are working, we will analyze a full set of CSF samples from two of the healthy young subjects that have been recruited for the ongoing NIH SILK-AB isoform study. Phase II of this grant will seek to further investigate the metabolism of Tau in populations relevant to Phase II clinical studies (mild cognitive impairment and age matched controls). In addition, we will test the reproducibility of the SISAQ-Tau assay and assess the combination of the SISAQ-Tau and SISAQ-AB assay for use as a potential diagnostic biomarker assay. PUBLIC HEALTH RELEVANCE: C2N will develop a novel mass spectrometer based method for measuring the metabolism and concentration of Tau in human cerebrospinal fluid. We will use this method to assist pharmaceutical companies understand the effects that their Tau targeting drugs impart in the human brain. Tau is a rapidly emerging and attractive drug target for the prevention or delay of Alzheimer's disease progression.